Anthracyclines of the daunorubicin group such as doxorubicin, carminomycin and aclacinomycin and their synthetic analogs are among the most widely employed agents in antitumoral therapy (F. Arcamone, Doxorubicin, Academic Press New York, 1981, pp. 12-25; A. Grein, Process Biochem., 16:34, 1981; T. Kaneko, Chimicaoggi May 11, 1988; C. E. Myers et al., "Biochemical mechanism of tumor cell kill" in Anthracycline and Anthracenedione-Based Anti-cancer Agents (Lown, J. W., ed.) Elsevier Amsterdam, pp. 527-569, 1988; J. W. Lown, Pharmac. Ther. 60:185-214, 1993). Anthracyclines of the daunorubicin group are naturally occurring compounds produced by various Streptomyces species and by Actinomyces carminata. Doxorubicin is mainly produced by strains of Streptomyces peucetius while daunorubicin is produced by many other Actinomycetes. In particular daunorubicin and doxorubicin are synthesized in S. peucetius ATCC 29050 and 27952 from malonic acid, propionic acid and glucose by the pathway summarized in Grein (Advan. Applied Microbiol. 32:203, 1987) and in Eckart and Wagner (J. Basic Microbiol. 28:137, 1988). Aklavinone (11-deoxy-.epsilon.-rhodomycinone), .epsilon.-rhodomycinone and carminomycin are established intermediates in this process. The final step in this pathway involves the hydroxylation of daunorubicin to doxorubicin by the DoxA enzyme ({U.S. Ser. No. 08/396,218, WO96/27014}; M. L. Dickens and W. R. Strohl, J. Bacteriol. 178:3389 (1996)), which is reported to occur only in S. peucetius.
13-Dihydrodaunorubicin may be an intermediate in the conversion of .epsilon.-rhodomycinone to daunorubicin via rhodomycin D (FIG. 1) according to Dickens et al. (J. Bacteriol. 179:2641 (1997)). Daunorubicin is bioconverted to (13S)-13-dihydrodaunorubicin when added to cultures of S. peucetius and some other Streptomycetes (N. Crespi-Perellino et al., Experientia, 38:1455, 1982; T. Oki et al., J. Antibiotics, 34:1229, 1981; G. Cassinelli et al., Gazz. Chim. Ital. 114:185,1984). It is not known whether the 13-dihydrodaunorubicin that may be an intermediate of daunorubicin and doxorubicin production in S. peucetius is identical to the (13S)-13-dihydrodaunorubicin formed by this bioconversion. Since these two compounds can differ in their C-13 stereochemistry, one diastereomer of 13-dihydrodaunorubicin might be a substrate for DoxA and the other one would not. In the latter case, C-13 reduction of daunorubicin would block its further oxidation to doxorubicin.
Several genes for daunorubicin and doxorubicin biosynthesis and resistance have been isolated from S. peucetius 29050 and 27952 by cloning experiments. The S. peucetius dnrU gene identified herein is a homolog of the Streptomyces sp. strain C5 gene ORF1 (syn. dauU) described by Dickens and Strohl (J. Bacteriol. 178:3389 (1996)). Since the predicted protein products of the dnrU and dauU genes resemble enzymes known to reduce ketone groups, the dnrU and dauU proteins may catalyze the reduction of daunorubicin, formed in vivo or added to cultures exogenously, to 13-dihydrodaunorubicin.
Daunorubicin is known to be converted to 4'-O-glycosides called baumycins in Streptomyces species (Y. Takahashi, H. Naganawa. T. Takeuchi, H. Umezawa, T. Komiyama, T. Oki and T. Inui. J. Antibiot. 30:622, 1977) thus decreasing also the amount of doxorubicin potentially obtainable through oxidation of daunorubicin. For recovering daunorubicin at the end of fermentation, baumycins are converted to daunorubicin by acid hydrolysis. However this process presents certain drawbacks in that the amount of doxorubicin thus produced is low and the process is complicated by the acid hydrolysis step. The present invention solves this problem by providing a mutated Streptomyces strain in which one of the genes responsible for the conversion of daunorubicin to baumycins has been insertionally inactivated.